1. Field of the Invention
This invention relates to a process for transformation of the alkane- and fatty acid-utilizing yeast of Candida tropicalis for industrial purposes. The transformation process described herein provides a means to introduce DNA fragments or sequences into host cells of C. tropicalis and allows C. tropicalis to be used as a host system for (1) gene expression and (2) polypeptide and protein production. Transformed yeast cells can be identified and selected by the methods of the present invention. Novel strains of C. tropicalis, vectors and subclones are provided. Novel yeast strains are used as hosts for introduction of recombinant DNA fragments.
The invention further relates to methods for stable transformation and maintenance of DNA in host cells. DNA is integrated into the genome by homologous recombination and both single and multiple tandem integrations are observed.
2. Description of the Related Art
There has been a continuing interest in developing important industrial eukaryotic micro-organisms for the production of a wide variety of products. In many cases, bacterial systems, such as E. coli or Bacillus are convenient and satisfactory, but for many purposes prokaryotic systems are inadequate. Prokaryotes do not provide processing of their peptides, such as glycosylation. In addition, prokaryotes do not efficiently secrete products. Products prepared in prokaryotes normally require cell lysis and then isolation of the proteins in the presence of large amounts of bacterial proteins and cellular debris. Prokaryotes have severe limitations for post-translational modifications and for production of products which can be isolated from the nutrient medium.
Yeast provide an attractive alternative to prokaryotes as host organisms. They can be used for large scale fermentations and are adaptable to continuous fermentation processing. In addition, they are not susceptible to phage infection and generally require only semi-sterile conditions. Conveniently, these cells may be immobilized for the production of metabolites and enzymes.
For some purposes, biochemical properties of yeast may be similar to higher organisms. For example, yeast may provide for glycosylation of polypeptide products. In addition, as eukaryotic organisms, the yeast codon preference may be similar to the codon preference of the organism from which the gene has been isolated. This may result in higher efficiencies of production of the desired protein. In addition, yeast are able to secrete proteins into the nutrient medium, which proteins can be readily recovered by conventional methods.
A number of yeast strains have been used as host organisms for recombinant gene expression. Lack of information about transformation conditions and appropriate vectors of C. tropicalis precluded development of a host/vector system for this yeast. Auxotrophic mutants were not available for C. tropicalis, thus preventing direct selection for transformants by auxotrophic complementation. The present invention overcomes such problems. The invention provides (1) a recombinant transformation system for C. tropicalis: (2) host strains, vectors and subclones for the transformation system; (3) methods for selection and identification of cells containing introduced DNA; (4) and methods to grow and maintain stably transformed cells. Further, genetically engineered C. tropicalis mutants may provide a means to screen chemical compounds for antifungal activity. A sensitive screening procedure for compounds toxic to this microorganism or related microorganisms (i.e., Candida albicans) would be of interest to the medical community.